Investigation of biological processes often requires the examination of those processes in cells, tissues and organs that comprise less than the entire organism. For many years these cells, tissues and organs have been separated from the organism and studied independently under conditions that support their survival in an ex vivo or in vitro mode. Typically, the cells, tissues or organs are removed from the organism and are maintained in a culture media that supports the survival and/or biological process being studied. Given the large diversity of cell, tissue and organ types, the formulation of culture medium that support their survival, growth and biological properties outside of the intact organism are not trivial. Many cells and tissues are difficult to maintain in culture for reasons that are not entirely understood. In addition, cells and tissues are often used in processes involved in the manufacturing of recombinant proteins, vaccines, virus stocks and other products in vitro which are key to biomedical research and biologics-based medicaments. Therefore, there is a need to identify conditions and culture media components that support the growth and survival of cells, tissue and organ cultures under in vitro and ex vivo conditions.
Such cell components include for example, albumin, transferrin, glutathione S-transferees, superoxide dismutase, lactoferrin, and growth factors.
Albumin is the most abundant protein found in the plasma. It is produced by the liver in mammals and functions in a variety of capacities. Albumin is a soluble, monomeric protein which comprises about one-half of the blood serum protein. Albumin functions primarily as a carrier protein for steroids, fatty acids, and thyroid hormones and plays a role in stabilizing extracellular fluid volume. Albumin is a globular unglycosylated serum protein of molecular weight 67,000 and contains five or six internal disulphide bonds. Albumin is synthesized as preproalbumin which has an N-terminal peptide that is removed before the nascent protein is released from the rough endoplasmic reticulum. The product, proalbumin, is in turn cleaved in the Golgi vesicles to produce the secreted albumin.
Albumin is essential for maintaining the osmotic pressure needed for proper distribution of body fluids between intravascular compartments and body tissues. It also acts as a plasma carrier by non-specifically binding several hydrophobic steroid hormones and as a transport protein for hemin and fatty acids. Bovine serum albumin (BSA) has long been used as a supplement in cell culture media as it is a component of fetal bovine serum (FBS) which is commonly added to a basal media at 1-20% total volume. BSA is a major component in a number of defined serum free media formulations since it is readily available in bulk, is relatively cheap, and can be purified to homogeneity relatively easily. Representative sources of albumin include for example, plasma derived from bovine, horse, pig and other mammalian species.
With the advent of the large scale production of recombinant proteins, vaccines and other products destined for human clinical use, stricter requirements on the formulations used in the production of those products have been instigated. Because of the threat of animal-derived materials harboring pathogens that may affect the safety of the products, many existing recombinant production processes have been modified such that all materials or culture components used in the entire process are devoid of animal-derived products. That is, the cell culture components cannot have been isolated or purified from whole animal sources. Therefore, the recombinant production of media supplements, as an alternative to the purification of these supplements directly from the whole animal is preferred. Accordingly, human and cell culture components from other species can be manufactured using recombinant means, using defined tissue culture media, and using highly characterized tissue culture cells, which are certified to be free of viruses and toxins.
One method of preparing recombinant protein based cell culture components is to engineer yeast or plants to over express the protein and then to purify the protein. Plant derived recombinant proteins are particularly attractive as a source of cell culture components for recombinant protein production of human proteins that are intended for therapeutic uses since there are no examples of plant viruses that can also infect humans.
There is currently a high demand for recombinant cell culture components to support the recombinant production of human therapeutic proteins, as well as to grow & differentiate stem cells, and with the continued success and huge potential of such products in the market, more effective ways of producing recombinant cell culture components is desirable. In particular, existing processes for the recombinant production of proteins and the growth and differentiation of stem cells are slow, expensive and arduous. In part these processes are limited by fundamental aspects relating to the rate of cell growth and viability of the recombinant host cells or stem cells respectively. Key aspects of these limitations include i) the ability to rapidly isolate and expand single cell clones from complex mixtures of cells, ii) the ability to promote rapid cell growth, particularly at low densities and in serum free media, iii) the ability to sustain cell growth and viability at very high densities in bioreactors, iv) the ability to cryopreserve and thaw cells and cell banks while maintaining high viability, v) the ability to grow and differentiate stem cell cultures effectively. Accordingly there is a need for improved media and culturing conditions that address these needs and enable the improved growth and viability of cells in culture. The supplements and methods of the invention, by improving the viability and rate of cell growth meet these needs and can also result in an improved yield and quality of recombinant product obtained from a mammalian cell culture production process.